Monoclonal antibody for diagnostic and therapeutic use in ovarian cancer

ABSTRACT

A method for the diagnosis and treatment of ovarian cancer. Tissue and body fluid samples are assayed for an antigen which specifically binds an anti-OSE antigen to diagnose ovarian cancer. Therapeutic agents coupled to anti-OSE antibodies, such as PS1 are specifically directed to ovarian cancer tissues.

This is a Continuation of Parent application Ser. No. 08/520,584 nowabandoned, filed on Aug. 29, 1995; which in turn is a Divisional ofapplication Ser. No. 08/326,053, filed Oct. 19, 1994 (now U.S. Pat. No.5,484,704); which in turn is a Continuation of application Ser. No.07/975,093, filed Nov. 12, 1992 (now abandoned).

FIELD OF THE INVENTION

The present invention relates to a method for the detection, monitoring,and treatment of ovarian cancer. More specifically, this inventionrelates to the use of a monoclonal antibody which specificallyrecognizes an ovarian antigen secreted by the ovarian surfaceepithelium.

BACKGROUND OF THE INVENTION

Cancer of the ovary is the second most common cancer of the femalereproductive organs and the fourth most common cause of cancer deathsamong American women. Because ovarian cancers are not readily detectableby diagnostic techniques (Siemens and Auersperg, 1988, J. CellularPhysiol., 134:347-356). Because diagnosis of carcinoma of the ovary isgenerally only possible when the disease has progressed to a late stageof development, it is one of the most lethal of the gynecologicalmalignancies.

There is a vital need for the identification of tumor markers which canbe used in the early detection of ovarian cancer, the monitoring ofcancer therapies, the immunodetection of ovarian tumors, and thedevelopment of probes for potential use in immunotherapy (Cantarow etal., 1981, Int. J. Radiation Oncol. Biol. Phys., 7:1095-1098). Althougha number of potential tumor markers have been evaluated including thecancer antigen 125 (Ca-125) nonspecificity of the antigens diminishtheir value as markers for primary ovarian cancer (Kudlacek et al.,1989, Gyn. Onc., 35:323-329; Rustin et al., 1989, J. Clin. Onc.,7:1667-1671; Sevelda et al., 1989, Am. J. Obstet. Gynecol.,161:1213-1216; Omar et al., 1989, Tumor Biol., 10:316-323).

Several monoclonal antibodies have recently been shown to react withovarian tumor associated antigens (Kenemans et al., 1989, Eur. J.Obstet. Gynecol. Repod. Biol., 29:207-218). These antibodies do notappear to be specific to a specific type of cancer (McDuffy, 1989, Ann.Clin. Biochen., 26:379-387). Many of these antibodies recognizedeterminants associated with high molecular weight glycoproteins relatedto mucins. Additional markers such as enzymes or activated cellularoncagenes or products of these genes have also been suggested for use asovarian cancer markers. These potential probes, however, have not beendetected in the blood and hence their use is confined to analysis oftumor tissues.

The majority, e.g., 80-90%, of ovarian cancers are thought to arise fromthe ovarian surface of epithelium (OSE). To date, little is known aboutthe structure and function of the cells of the ovarian surfaceepithelium. It is known that the surface epithelium is a highly dynamictissue which undergoes morphogenic changes. The OSE also has significantproliferative properties, as it it must proliferate rapidly to cover theovulatory site after ovulation of the ova. Morphological andhistochemical studies suggest that the OSE has secretory, endocytoticand transport functions which are hormonally controlled (Blaustein andLee, 1979, Oncol. 8:34-43; Nicosia and Johnson, 1983, Int. J. Gynecol.Pathol., 3:249-260; Papadaki and Beilby, 1971, J. Cell Sci., 8:445-464;Anderson et al., 1976, J. Morphol., 150:135-164).

There is a vital need for an ovarian cancer marker which canspecifically detect-ovarian cancer, which marker can be used todiagnose, monitor and identify ovarian cancers and which may be used inspecific immunotherapy.

SUMMARY OF THE INVENTION

The present invention provides a method for the specific detection,monitoring, and/or treatment of ovarian cancer. In the method of thepresent invention, an in vitro assay employs antibodies whichspecifically recognize an antigen of the ovarian surface epithelium(OSE) to determine the presence and amounts of OSE antigen in tissues orbody fluids of patients. Such assay permits detection of ovarian cancer,monitoring of ovarian cancers and specific identification of ovariancancer type. Anti-OSE antibodies are also used to specifically targettherapeutic agents to ovarian cancer cells.

For diagnosing and classifying ovarian cancer types, ovarian cancertissues are probed with an anti-OSE antibody, e.g., byimmunohistochemical methods. For detection and monitoring of ovariancancer in body fluids, the anti-OSE antibodies are preferably used inimmunoassays to detect a secreted OSE antigen. For therapeutic purposes,anti-OSE antibodies conjugated to cytotoxic agents are administered fortargeted delivery to OSE antigens present in the ovarian cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows localization of PS1 antigen in OSE of cynamologous monkeys(FIG. 1A), baboons (FIG. 1B), rabbit (FIG. 1C) and human (FIG. 1D). FIG.1E shows a section of human ovary probed with control, anti-myelomaantibody. Arrows indicate ovarian surface epithelium.

FIG. 2 shows histological sections of paraffin embedded human ovariantumors.

FIG. 2A shows sample 13022 stained with PAS.

FIG. 2B shows sample 13022 reacted with PS1 antibody.

FIG. 2C shows sample 9010 stained with PAS.

FIG. 2D shows sample 9010 reacted with PS1 antibody.

FIG. 2E shows sample 10035 stained with PAS.

FIG. 2F shows sample 10035 reacted with PS1 antibody.

FIG. 2G shows sample 13017 stained with PAS.

FIG. 2H shows sample 13017 reacted with PS1 antibody.

FIG. 2I shows sample 9160 stained with PAS.

FIG. 2J shows sample 9160 stained with PS1 antibody.

FIG. 2K shows sample 10880 stained with PAS.

FIG. 2L shows sample 10880 reacted with PS1 antibody.

FIG. 2M shows sample 9219 stained with PAS.

FIG. 2N shows sample 9219 reacted with PS1 antibody.

FIG. 3 shows histological sections of paraffin embedded normal humanovaries.

FIG. 3A shows sample 13053 stained with PAS.

FIG. 3B shows sample 13053 reacted with PS1 antibody.

FIG. 3C shows sample 13012 stained with PAS.

FIG. 3D shows sample 13012 reacted with PS1 antibody.

FIG. 4 shows histological sections of a paraffin embedded Brennerstumor.

FIG. 4A shows sample 13044 stained with PAS.

FIG. 4B shows sample 13044 reacted with PS1 antibody.

FIG. 5 shows an autoradiograph of proteins radiolabeled with ³⁵S-Translabel, secreted by rabbit OSE in vitro, immunoprecipitated withanti-PS1 antibody, and electrophoresed in 7.5% polyacrylamide. Lanes 1-3contain OSE secretory proteins.

DETAILED DESCRIPTION OF THE INVENTION

In the method of the present invention, antibodies which specificallyrecognize antigens of the ovarian surface epithelium are used asdiagnostic or therapeutic tools to determine the presence and amounts ofOSE antigen in the tissues or blood of patients to diagnose, identify,and monitor ovarian cancer. In an alternative embodiment anti-OSEantibodies are used to specifically target therapeutic agents to tissuesexpressing OSE antigens, e.g., ovarian cancer tissues.

The antibodies used in the method invention specifically bind antigensof the ovarian surface epithelium, and also recognize antigens presentin the cell surface membranes and preferably for diagnosis, antigenssecreted by these epithelial cells.

For diagnosing and classifying ovarian cancer types, the anti-OSEantibodies preferably bind ovarian cancer tissues. For detection ofovarian cancer in body fluids such as blood, peritoneal fluid, tissuefluid, and the like, the anti-OSE antibodies preferably bind antigenssecreted by the OSE and by the ovarian cancer cells. For therapeuticpurposes, the anti-OSE antibodies preferably bind antigens associatedwith the cell surface membranes of the ovarian cancer, and therebydeliver therapeutic agents to the tumor.

Antibodies useful in the present invention may be polyclonal,monoclonal, or recombinant antibodies, and are prepared by methodsgenerally known in the art. See, for example, Dunbar, 1987, TwoDimensional Gel Electrophoresis and Immunological Techniques, PlenumPress, New York, and Larrick and Fry, 1991, Human Antibodies andHybridomas, 2:172-189.

Antigens which may be used to produce antibodies useful in the methodinvention are those present in the ovarian surface epithelium (OSE) andthose antigens secreted by the OSE. The antigens may be, for example, aprotein, a glycoprotein, or a carbohydrate moiety.

Antibodies to be used in the method invention are those whichspecifically bind normal OSE and those which specifically bind anantigen secreted by normal OSE, and also specifically bind ovariancancer cells or antigens secreted by ovarian cancer cells.

The phrases "specifically binds" and "having specificity for" are meantto include binding of the anti-OSE antibody to ovarian tissues and toovarian cancer cells with little or no binding to non-ovarian tissues orcancers of non-ovarian origin.

For example, the monoclonal antibody PS1 described by Tivnons and Dunbar(1988, in Mathur and Fredericks, eds., Perspectives inImmunoreproduction: Conception and Contraception, Hemisphere Press, NewYork, p. 242) has surprisingly been found to recognize an antigenpresent in the ovarian surface epithelium of cynomolgus monkey, baboon,rabbit, and human ovarian tissues. PS1 also binds an antigen secreted byovarian cell surface epithelium, in vitro, and specifically recognizesan ovarian cancer antigen.

The PS1 antibody has been previously described as recognizing a ovarianzona pellucida antigen, and has been characterized as recognizing acarbohydrate constituent of this antigen (Timmons et al., 1987, Biol.Reprod., 36:1275). The-PS1 monoclonal antibody has been found tolocalize not only to the oocyte and its surrounding extracellular matrixbut also in the secretory, apical portion of the ovarian surfaceepithelium.

PS1 specifically binds ovarian tumor tissue and can be used todistinguish ovarian cancer cells from non-carcinogenic tissue fordiagnostic purposes and for the classification of tumor types based uponhistological sections and immunohistochemical assays.

When the antibody PS1 is used in immunohistochemical assays of humancancer tissues, the antibody specifically binds throughout the ovariancancer tissue while showing no significant binding to non-ovarian cancertypes. Normal ovaries show binding of the antibody in the remnants ofthe OSE.

The hybridoma cell line which produces monoclonal antibody PS1 wasdeposited under the provisions of the Budapest Treaty and accepted bythe American Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209, on Oct. 8, 1987, and given the accessionnumber HB9565.

Metastatic ovarian cancer present in non-ovarian tissues may beidentified using the method of the present invention. Thus, antibodieswhich specifically recognize antigens of the OSE are used to identifyovarian cancer tissue. The identified ovarian cancer tissue may bemetastatic ovarian cancer cells present in non-ovarian tissues.

Preferably, the tissue to be assayed will be obtained by surgicalprocedures, e.g., biopsy. The excised tissue will be assayed for thepresence of an antigen which recognizes an anti-OSE antibody asdescribed above, e.g., PS1, by methods generally known in the art, e.g.,imunohistochemistry. The tissue may be fixed or frozen to permithistological sectioning, and may be stained prior to incubation with theantibody. The antibody may be labeled, for example with a dye orfluorescent label, chemical, heavy metal or radioactive marker to permitthe detection and localization of the antibody in the assayed tissue.Alternatively, other immunological assays known to those of skill in theart may be employed which permit detection of antibody binding to theexcised tissue.

Because the PS1 antigen is a carbohydrate moiety, and has now been foundto be a secretory antigen in the oocyte, secretion of the PS1-recognizedantigen by OSE provides a specific ovarian cancer marker for detectionin body fluids of patients presenting with proliferation of this celltype, e.g., ovarian cancer. Additional antigens secreted by the OSE maybe used to prepare antibodies which, like PS1, may be used to diagnoseovarian cancer by immunoassay of tissue and body fluids for the presenceof an antigen present in or secreted by ovarian cancer cells andrecognized by the anti-OSE antibody.

Body fluids such as blood and fluid blood components, peritoneal fluid,tissue and the like may be assayed for the presence and amount ofantigen which binds anti-OSE antibodies. The assay may be performed bymethods routinely used by those of skill in the art. Generally, a sampleof body fluid is added to an assay mixture containing the antibody and amarker system for detection of antigen-bound antibody. Examples of suchimmunoassay systems are radioimmunoassays (RIA), enzyme-linkedimmunoassays (ELISA), immobilized immunoassays, and the like.

Therapeutic agents useful in the treatment of ovarian cancer may beconjugated to the anti-OSE antibodies by methods known to those of skillin the art. Examples of such therapeutic agents include radioisotopessuch as ¹³¹ I or ⁶⁷ Cu and cytotoxic agents such as ricin or diphtheriatoxin. ¹³¹ I can be covalently bound to antibody molecules by methodsknown in the art, e.g., Iodogen® reactions. ⁶⁷ Cu may be attached by wayof an appropriate metal chelator bound to the antibody. The toxins maybe conjugated to antibodies by agents such as glutaraldehyde orcarbodiimide.

Administration of the conjugated therapeutic agent-antibody may be byvarious means known in the art, but generally will be by injection,systemic or intraperitoneal and in a pharmaceutically acceptablecarrier. A useful therapeutic dose will vary with the particulartherapeutic agent used, the particular cancer type and history, and thespecific considerations of the patient to be treated. A physicianadministering the agent will know to calculate the effective therapeuticdose, which dose will be effective in reducing or eliminating the tumortissue without compromising significantly normal tissues of the patient.

The nature of the invention can be further understood by reference tothe following examples.

EXAMPLE 1 IDENTIFICATION OF PS1 DETERMINANT IN OVARIAN SURFACEEPITHELIUM

Initial immunohistochemical localization studies demonstrated that thePS1 monoclonal antibody localized not only to the oocyte and surroundingextracellular matrix of the zona pellucida but also localizedintensively in the secretory, apical portion of the ovarian surfaceepithelium.

Human and cat ovarian tissue was embedded and immediately frozen in OCTcompound (Miles, Elkhart, Ind.). Cryostat sections, 4-8 μm were preparedusing a Minotome/microtome cryostat, and the sections were fixed in 100%methanol for 10 minutes, blocked in TRIS-buffered saline (TBS), pH 7.5,containing 2.5% w/v Brij-35 (SIGMA, St. Louis, Mo.) and 6% w/v Carnationnon-fat dried milk. Blocking-proceeded for thirty minutes at 37° C. in amoist chamber and was followed by brief rinsing in TBS/Brij.

The blocked sections were next incubated with the monoclonal antibodyPS1 in a moist chamber for 30 minutes at 37° C. PS1 antibody wasprepared as described in Timmons and Dunbar, 1988, in Mathur andFredericks, eds., Perspectives in Immunoreproduction: Conception andContraception, Hemisphere Press, New York, p. 242. Control sections weretreated with IgG isolated from myeloma cell acetous fluid. Theincubation was followed by rinsing in TBS/Brij and application ofsecondary antibody under the same conditions. The secondary antibody,peroxidase-labeled rabbit anti-mouse IgG (Dako, Santa Barbara, Calif.)was diluted 1:50 in blocking solution before application and wasfollowed by washing in TBS and subsequent treatment with a solution of3,3'-diaminobenzidine (Fisher Scientific, Fairlawn, N.J.) consisting of50 mg freebase/100 ml TBS containing 0.0031% hydrogen peroxide at roomtemperature.

Rabbit and cynomolgus monkey ovarian tissue was similarly prepared andtreated. Morphology was improved by finely mincing the rabbit tissues,fixing in 1% glutaraldehyde for one hour at room temperature, washingseveral times in chilled PBS, dehydration, and imbedding in Eponaccording to the manufacturer's instructions. Plastic embedment wasfollowed by sectioning at 1-3 μm on a Mt-2 ultra microtome (Sorvall).Mounted sections were briefly etched (15 minutes) in sodiumethoxide/ethanol (1:1) and light trypsination (2.5% sucrose treatmentfollowed by incubation in 0.0001% trypsin for 30 minutes at 37° C.). Theblocking, antibody treatment, and signal development were performed asdescribed above for human and cat ovarian tissue.

As shown in FIGS. 1A-1E, the PS1 antigenic determinant was localized inthe ovarian surface epithelium of cynomolgus monkey (FIG. 11A), baboon(FIG. 1B), rabbit (FIG. 1C), and human (FIG. 1D) with intenselocalization on the apical portion of OSE (arrows). In contrast, thecontrol myeloma antibody did not recognize human surface epithelium(FIG. 1E).

EXAMPLE 2 IDENTIFICATION OF PS1 DETERMINANT IN HUMAN OVARIAN CANCERTISSUE

Human ovarian tissue, both normal controls and tumor tissues wereobtained as fixed sections embedded in paraffin from the Department ofPathology, M.D. Anderson Hospital, Houston, Tex. Immunohistochemicalassay of the sections was performed as follows:

Paraffin was first removed and the sections rehydrated by six (6) minutewashes, in each of Xylene (twice), 100% ethanol (twice), 95% ethanol,85% ethanol, 70% ethanol, 50% ethanol, and distilled, deionized water.The sections were next incubated 15-20 minutes in 3% hydrogen peroxidein methanol and washed 3 times in PBS containing 0.1% BSA, 5 minutes perwash. The washed sections were next blocked by incubating 20 minuteswith normal rabbit serum (diluted 1:500) in a humidified chamber at roomtemperature, and washed 3 times in PBS containing 0,1% BSA, 5 minutesper wash. The blocked sections were then incubated with PS1 IgG (1:500)or control antibody, IgG derived from ascites fluid of mice injectedwith p3U1 myeloma cells (Drell & Dunbar (1984) Diolopy of Reproduction,30:445-457) (1:500) for two hours at room temperature, or overnight at4° C., followed by three PBS washes as described above. Second antibodywas applied by incubating with biotinylated rabbit anti-mouse IgG(Vectastain, Vector Laboratories, Burlingame, Calif.) for thirty (30)minutes at room temperature, followed by three PBS washes as describedabove. The sections were next incubated 30 minutes at room temperaturewith the avidin biotinylated horseradish/peroxidase reagent (ABC)(Vectastain) and washed three times in PBS as described above.

To stain the sections, 2 drops of 3% H₂ O₂ was added to diaminobenzldinetetrahydrochloride DAB solution (3-5 mg/10 ml 0.1 M TRIS) iimediatelybefore use. The sections were incubated with the prepared DAB solutionfor 5-10 minutes, washed twice in dd H₂ O, and coverslips applied.

The assayed sections were then viewed by light microscopy. Sections of"normal" human ovarian tissue (ovarian tissue having no gross pathologyas evaluated by M.D. Anderson pathologists) was compared with sectionsof tissue surgically removed from cancer patients.

As shown in FIGS. 2, 3 and 4, the monoclonal antibody PS1 specificallyrecognized an antigen present in human ovarian tumors (FIGS. 2A-2N). Incontrast, antibody specific antigen was identified only in the remnantovarian cell surface epithelium of normal, adult ovarian tissues (FIGS.3A-3D). FIGS. 4A and B show histological sections of a Brenner's tumor.This tumor is generally considered to be non-malignant and does notreact with the PS1 antibody, which specifically identifies ovarianmalignancies derived from OSE. Histological data for these andadditional human tissue samples is summarized in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    SAMPLE                      PS1 PRESENCE.sup.b                                                             NO. TISSUE DIAGNOSIS (Localization)              __________________________________________________________________________    13012                                                                              OVARY     NORMAL       RESIDUAL OSE                                           ONLY (IN CRYPTS)                                                           13057 OVARY NORMAL + Residual OSE                                             13053 OVARY NORMAL -                                                          13018 OVARY NORMAL -                                                          13004 OVARY NORMAL -                                                          13065 OVARY NORMAL -                                                          9213 OVARY NORMAL -                                                           13017 OVARY ADENOCARCINOMA OF +                                                 ENDOMETRIUM                                                                 13078 OVARIAN TUMOR PAPILLARY SEROUS ++                                         CARCINOMA                                                                   9129 OVARIAN TUMOR BRENNER'S MUCINOUS + (FEW                                    TUMOR METASTATIC ISOLATED                                                     ADENOCARCINOMA OF EPITHELIAL                                                  LIVER CELLS)                                                                13022 OVARIAN TUMOR PAPILLARY SEROUS +++                                      9208 OVARIAN TUMOR TERATOMA TUMOR ++                                          13027 OVARIAN TUMOR SEROUS CYST +                                             9010 OVARIAN TUMOR SEROUS METASTATIC ++                                       13044 OVARIAN TUMOR BRENNER'S TUMOR -                                         13028 OVARIAN TUMOR GRANULOSA CELL +++                                          TUMOR (RESEMBLES                                                              PAPILLARY SEROUS)                                                           10880 OVARIAN TUMOR SEROUS CARCINOMA +++                                      1068 OVARIAN TUMOR METASTATIC +/- (SMALL                                        TRANSITIONAL POCKETS)                                                       9160 OVARIAN TUMOR ADENOCARCINOMA OF +++                                        ENDOMETRIUM                                                                 13023 OVARIAN TUMOR MATURE CYSTIC -                                             TERATOMA                                                                    9219 OVARIAN TUMOR ENDOMETRIUM ++                                               CARCINOMA OF OVARY                                                            WITH METASTATIC                                                               ADENOCARCINOMA                                                              10890 OVARIAN TUMOR MUCINOUS CYSTIC ++                                        4703 OVARIAN TUMOR PAPILLARY SEROUS ++                                        10035 OVARIAN TUMOR PAPILLARY SEROUS ++                                     __________________________________________________________________________     .sup.a Classification and tumor diagnosis by pathologists from M.D.           Anderson Cancer Institute (Houston, TX)                                       .sup.b Monoclonal antibody PS1 localization: + = few cells but distinct;      ++ = more general localization; +++ = predominant localization.          

EXAMPLE 3 SPECIFICITY OF ANTI-OSE ANTIBODY TO OVARIAN CANCER TISSUE

Tumors were obtained and analyzed as described for Example 2 for bindingof the anti-OSE antibody, PS1. As shown in Table 2, non-ovarian tumortissue did not react positively with the PS1 antibody. In contrast, anomentum tumor taken from a patient diagnosed with metasticadenocarcinona of the ovary was highly reactive with the PS1 antibody.These results demonstrate the use of anti-OSE antibodies to characterizemetastatic tumors as having an ovarian origin.

                  TABLE 2                                                         ______________________________________                                                                    REACTIVITY                                          SAMPLE NO. TUMOR DESCRIPTION WITH ANTI-PS1                                  ______________________________________                                        UB 2102     Uterine tumor   Negative                                            PN 2376 Uterine tumor Negative                                                SB 0379 Perivaginal tumor Negative                                            SS 0688 Vulva tumor Negative                                                  VM 9758 Omentum* tumor +++                                                  ______________________________________                                         *Omentum tumor from patient having metastatic adenocarcinoma of ovary    

EXAMPLE 4 PS1 BINDS AN ANTIGEN SECRETED BY OSE

Both ovaries were removed from a mature (>14 weeks) New Zealand Whiterabbit, trimmed, and placed into warm PBS, pH 7.2. The ovaries wererinsed three times in Hank's Balanced Salt Solution (HBSS) (GIBCO) andplaced into a 25 ml Erlenmeyer flask containing 5 ml Dulbecco's ModifiedEagle Media (DMEM) without methionine (Met-free medium) (GIBCO), 37° C.The flask was gassed for 15 minutes in a 37° C. incubator at 5% CO₂,then stoppered and moved to a 37° C. shaker-incubator and swirled gentlyat 70-80 rpm for 45 minutes.

The ovaries were next removed to a second Erlenmeyer flask containingmedium as described above but also containing 100 uCi of ³⁵ S-Translabel(Met:Cys 14:3) (ICN, Irvine, Calif.). The 15 minute gassing, 45 minuteswirling incubations described above were repeated four times. Themedium was removed and concentrated five times using a Centriconmicroconcentrator having a 10,000 MW cut-off and centrifuging at5000-6000 rpm at 4° C.

The resulting concentrated medium was assayed by immunoprecipitationwith the monoclonal antibody, PS1. To eliminate non-specific binding, 25ul of normal rabbit serum was added to 200 ul of the concentratedmedium, and the samples were agitated on a platform rocker for one hourat room temperature. To this mixture was added 50 ul of a 10% suspensionof Protein A Sepharose CL-4B (Pharmacia) suspended in NP-NET buffer. Thesamples were rocked for two hours at room temperature, or overnight at4° C., then centrifuged at 12,000-13,000 rpm for ten minutes, and thesupernatant removed.

To the adsorption-treated supernatant was added 100 ul of PS1 antibody(1.6 mg/ml protein) and the samples were rocked for two hours at roomtemperature (or overnight at 4° C.). After incubation with PS1, 50 ul ofaffinity purified rabbit anti-mouse IgG (1 mg/ml protein) was added andthe samples were again rocked for one hour at room temperature. A volumeof 100 ul of the 10% suspension of Protein A-Sepharose in NP-NETdescribed above was added to each sample which was then vortexed brieflyand rocked for two hours at room temperature or overnight at 4° C. Thesamples were then centrifuged at 12,000-13,000 rpm for ten minutes andthe supernatant removed. The pellet containing PS1-bound ³⁵ S-labeledproteins was washed twice in NP-NET buffer and analyzed bySDS-polyacrylamide gel electrophoresis (PAGE).

To each sample pellet was added freshly made SDS solubilization buffer(0.0625 M Trizma base containing 10% glycerol and 2% SDS to which 2-5%beta-mercaptoethanol was added immediately prior to use). The mixturewas heated in a boiling water bath for ten minutes, cooled to roomtemperature, centrifuged at 12,000-13,000 rpm for ten minutes and thesupernatant removed for electrophoresis.

One dimensional PAGE was performed using conventional procedures. A 10%polyacrylamide gel was prepared, and samples added to lanes andelectrophoresed. The resulting gel was stained with Coomassie bluestain, destained in ethanol/acetic acid, and treated with 1X EN³ HANCEreagent (Dupont) for one hour with gentle agitation. The reagent wasdiscarded and the gel was washed with deionized water for 30 minutes.The washed gel was dried and placed against X-ray film for theproduction of autoradiographs. Development of the film overnight at -70°C. was generally sufficient for the production of a strong signal.

The resultant autoradiograph is shown in FIG. 5. Lanes 1-3 containedproteins immunoprecipitated from the conditioned medium of the ovariesincubated as described above. These proteins may be presumed to besecreted predominantly by the ovarian surface epithelium cells and notother ovarian cell types because of the short incubation period. Onlycells at the surface of the ovary are expected to take up the labeledamino acids, incorporate label into protein, and release labeled proteininto the culture medium in such a short time. The OSE cells are thoseavailable to the label on the surface of the ovary. Lane 4 containedmolecular weight markers. Lane 5 contained cytoplasm of isolated OSEcells and Lane 6 plasma membranes isolated from OSE after labeling asdescribed above.

EXAMPLE 5 IDENTIFICATION OF ANTIGENS SECRETED BY OSE

Antigens secreted by the OSE are useful for the production of antibodiesfor the detection of tissue and secreted antigens conserved in ovariantumor cells. Such antigens may be prepared and screened, for example, bythe following procedure.

OSE is isolated from the ovaries of a mammal, e.g, rabbit, as adaptedfrom Nicosia et.al, International Journal of Gyn. Path., 1984,3:348-360. Ovaries are rinsed three times briefly in HBSS at 37° C. andplaced into a 50 ml plastic centrifuge tube containing 10 mldissociation solution (10 ml Medium 199 containing 3000 units ofcollagenase, 25 units/ml penicillin and 50 ug/ml streptomycin, the finalmixture filtered through a 0.22 u filter before use and at 37° C.). Thesample is gassed for 15 minutes at 5% CO₂ and 37° C., then stoppered andmoved to a 37° C. shaker-incubator and swirled gently (70-80 rpm) for 45minutes.

After dissociation, the contents of the tube are poured into a plasticpetri dish and the ovaries gently lifted into a 15 ml plastic centrifugetube containing 10 ml Medium 199 at 37° C. The solution is aspiratedgently five times with a Pasteur pipette, and then vortexed. Thesolution is then gently aspirated again five times, and the contentspoured onto a watch glass. The ovary is immobilized with a needle placedthrough its long axis and the surface of the ovary is gently scrapedwith a scalpel blade, releasing the OSE into the solution.

The ovaries are then removed and the solution containing the OSE isplaced into a 15 ml plastic centrifuge tube and centrifuged at 1400 rpmfor five minutes. The supernatant is discarded and the pelletresuspended in 3 ml Medium 199 at 37° C. The solution is then layeredonto a 10 ml "unit gradient" (5% BSA in Medium 199, pH 7.2, sterilefiltered) and allowed to sit undisturbed for twenty minutes.

All but the lower 3 ml of the "gradient" is pipetted off and discarded.The remaining 3 ml is centrifuged at 1400 rpm for 5 minutes, theresulting supernatant is discarded, and the pellet washed twice with 10ml HBSS at room temperature, and again centrifuged.

The OSE cells are then cultured by means known in the art, for example,using conditions as described in Maresh et al, 1990, Biol. Reprod.,43:965-976. The conditioned medium is then collected from the OSEcultures and proteins concentrated, for example, using the diatomaceousearth procedure described by Dunbar, 1987, in Two DimensionalElectrophoresis and Immunological and Immunological Techniques, p.223-225.

The OSE secreted proteins are analyzed by two-dimensional gelelectrophoresis, by methods known in the art. See, for example, Dunbar,1987, Two Dimensional Electrophoresis and Immunological Techniques,Plenum Press, New York. Proteins detected by staining, i.e., Coomassieblue or silver stain, are isolated from the polyacrylamide gel usingprocedures known in the art (see Dunbar, 1987) and used to injectanimals to prepare polyclonal and monoclonal antibodies (see Dunbar,1987).

The PS1 antibody can be used to immunoprecipitate secreted OSE proteins,as described in Example 3 above. Proteins identified using thisprocedure may be separated by two-dimensional PAGE and purified for useas antigens to prepare anti-OSE antibodies.

Efficacy of anti-OSE antibodies prepared as described herein for utilityas ovarian cancer markers can be tested by the antibody's recognition ofovarian tumor tissue using the immunohistochemical procedures describedin Example 2, as well as identification of antigens secreted by humanovarian cancer cell lines. Secretion of antigen into the body fluids andor circulation in the blood may be evaluated using conventionalimmunoassay techniques, e.g., competitive ELISA procedures.

EXAMPLE 6 TARGETING OF THERAPEUTIC AGENTS

Therapeutic agents may be conjugated to anti-OSE antibodies for targeteddelivery to ovarian cancer tissue. For example, cytotoxic agentsincluding radioisotopes and cytotoxins such as ricin may be conjugatedby means known in the art to anti-OSE antibodies.

The specific binding of the anti-OSE antibody to ovarian cancer cellsenables specific delivery of the agent to the cancer tissue.

I claim:
 1. A method of targeted delivery of a therapeutic agent toovarian cancer tissue comprising the steps of:conjugating a therapeuticagent to PS1 antibody secreted by hybridoma cells having ATCC Accessionnumber HB9566; and administering the conjugated antibody to a patient.